The course of hepatitis B virus (HBV) infection in humans can be monitored by following the appearance and disappearance of certain components in plasma or serum of infected subjects. There appear to be three major HBV antigenic proteins: HBs, the envelope protein or surface antigen; HBc, or core antigen; and HBe antigen, which appears to be a processed product of the core. Antibodies are formed to all three of these antigens. The core antigen, HBc, does not appear as such in the plasma of infected subjects, however, its processed product HBe, along with HBs, are found in the plasma within several months after infection, and then effectively disappear. Antibodies reactive with core (anti-HBc) begin to appear about two months after infection and concentrations in plasma of antibodies reactive with HBe antigen (anti-HBe) peak around 4 to 5 months after infection. Anti-HBs antibody titers rise more or less concomitantly with the diminution of titers of anti-HBe. Thus, by assessing the levels of all five plasma-borne components, HBs, HBe, anti-HBs, anti-HBe, and anti-HBc, the status of the infection can be assessed. Commercially available assay kits provide tests for all of these markers.
Although HBc antigenic activity is not detected in plasma of infected individuals, particles containing the core antigen can be isolated. It has been known for over ten years that HBe antigen is released from these core particles by treatment with pronase, with pronase and 2-mercaptoethanol, with sodium dodecyl sulfate and 2-mercaptoethanol, or through disruption by sonication and treatment with chaotropic agents. Therefore, it is assumed that HBe is some sort of processed product of HBc, and that when HBc is produced in mammalian systems, its antigenic characteristics are converted to those of HBe. However, it has appeared that in order to maintain the anti-HBe characteristics of the processed antigen, denaturing conditions must be maintained. If the "processed" protein is put back into isotonic solution, it reassumes the antigenic properties of HBc.
As proteolytic cleavage appears to be involved in converting HBc to HBe, it is also known that the HBe antigen (or mixture of antigens) is a shorter molecular weight form of HBc. The native coding sequence and the deduced amino acid sequence for HBc have been known for some time (see, for example, U.S. Pat. No. 4,710,463 to Biogen).
U.S. Pat. Nos. 4,758,507 and 4,563,423, both assigned to Biogen, describe the recombinant production of putative HBe. Briefly, the methods involve recombinant production of HBc in bacteria and subsequent treatment with reagents to convert the HBc product to HBe. In illustrative embodiments, HBc of about 1% purity is treated either with 0.1% pronase or with 0.1% pronase and 0.1% mercaptoethanol. Alteratively, 1% SDS and 10 mM 2-mercaptoethanol are used. It is further suggested that the HBe recombinant protein could be prepared by chewing back the HBc gene to an appropriate but unspecified location to generate HBe peptide. However, the HBe produced by these methods, even the putatively shortened form, require the presence of denaturing agents to maintain HBe antigenicity.
European application No. 87117370.4 (Publication No. 0,272,483) assigned to Abbott Laboratories describes recombinant production of HBe from a C-terminal deleted HBc gene and maintenance of HBe antigenic characteristics by treatment with and storage in guanidine. Again, removal of the chaotropic agent results in resumption of HBc rather than HBe antigenic characteristics.
There thus exists a need for a method of maintaining the antigenic characteristics of HBe without the use of denaturing or chaotropic agents. The present invention satisfies this need and provides related advantages as well.